BIODIVERSITY GENOMICS EUROPE WP4
High Mountain Systems -
Arthropod sampling with Malaise traps
SOP
First version: 23 June 2023
Last version: 07 November 2024
Laura Nájera-Cortazar [BIOPOLIS-CIBIO]
Sónia Ferreira [BIOPOLIS-CIBIO]
Vanessa Mata [BIOPOLIS-CIBIO]
Pedro Beja [BIOPOLIS-CIBIO]
Associação Biopolis - CIBIO Centro de Investigação em Biodiversidade e Recursos
Genéticos [BIOPOLIS - CIBIO]
BIODIVERSITY GENOMICS EUROPE
receives funding from the European Union's Horizon Europe Research and Innovation Action.
https://biodiversitygenomics.eu/
2 | 4.4 HMS Sampling protocol / PlutoF Go
Table of contents
Introduction 2
Sampling Design 3
Permits and documentation 5
Before starting 6
Setting Malaise traps and sample collection 8
Shipment 11
Registering samples in PlutoF Go app 12
Acknowledgements 15
References 15
Useful contact 15
Introduction
The Biodiversity Genomics Europe (BGE) Consortium has the overriding aim of accelerating the
use of genomic science to enhance understanding of biodiversity, monitor biodiversity change,
and guide interventions to address its decline. The objective is to establish functioning
biodiversity genomics networks, data generation and pipelines to characterize biodiversity, and
to improve management intervention and biomonitoring programs by practical application of
genomic tools.
Arthropod biodiversity is mostly unknown and highly understudied, despite its importance for
ecosystem functionality (Outhwaite et al., 2022; Srivathsan et al., 2023). Studies have identified
that insect biodiversity changes are mostly driven by intensive human land-use and climate
change (Outhwaite et al., 2022), but how these factors interact under different systems is
unclear. Identifying the drivers of arthropod biodiversity loss requires allocating resources for
species discovery, and understanding how community composition is shaped worldwide
(Srivathsan et al., 2023). The use of DNA barcoding (Herbert et al., 2003) and metabarcoding
(Taberlet et al., 2012) techniques, represent an effective way to identify diversity by analyzing
bulk samples of specimens (Young and Herbert, 2022).
Within the BGE scope, one of the objectives is to assess pan-European patterns of species
diversity and community composition in key systems, establishing baseline sampling in
mountain ranges across Europe to track biodiversity shifts associated with climate change. The
“High Mountain Systems - Arthropods” case study is designed to evaluate how arthropod and
pollinator communities change (species diversity and composition) along altitudinal gradients in
selected mountain systems in Europe, showcasing the use of DNA-based tools.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Sampling Design
For this Case Study, sampling will be carried out in mountain systems of different European
countries representing different biogeographic settings. Malaise traps (Figure 1) will be placed in
each mountain for sampling arthropods at different altitudinal gradients. A Malaise trap is a
tent-like trap that will canalize arthropods to go to the top of the trap, for them to fall into a
collection tube attached to the top central section of the trap, containing 96% ethanol. For the
BGE, we are following the procedures stated in the Global Malaise Trap program, where you
can find further information about the program.
Figure 1. Scheme of a Townes Style Malaise trap used in the present study and its approximate
dimensions (left); and example of a Malaise trap set in Serra da Estrela, Portugal (right).
For each Mountain range, an altitudinal gradient will be sampled, setting Malaise traps at five
elevation sites (Figure 2). The selection of these sites should be done according to each
mountain system’s characteristics, i.e. including representative vegetation of each gradient.
Sampling should start at the highest point accessible (or feasible to access every week), and
then go down at regular intervals. A pair of Malaise traps will be placed per site/elevation, at
least 50 m from each other. Malaise traps will operate for a total of 20 weeks, with samples
collected from each trap at weekly intervals. From each pair of traps, one sample will be used
for characterising the arthropod community using DNA metabarcoding; and the other sample
will be used as a backup to the former, in case of damage or other operational issue, or as a
source of specimens for morphological studies and to be sequenced as fresh material aiming to
complete the DNA barcoding reference database. Detailed information about how to set the
Malaise traps is described in page 8.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Sample tubes must be collected every week during 20 weeks, trying to be as consistent as
possible to collect the samples on the same day of the week during all the sampling time. This
will give a total of 200 bulk samples per mountain range that will be sent for DNA metabarcoding
analyses. Please note that once removed from the Malaise trap, sample tubes should not be
reopened for any reason to avoid contamination. Store the samples safely at room
temperature and avoid light exposure.
Figure 2. Example of the altitudinal gradient sampling scheme.
The PlutoF platform (Kessy et al. 2010) will be used as a workbench for processing the
metadata. It includes a mobile app, PlutoF Go, that will be used for data entry during fieldwork.
A set of 250 stickers with unique QR codes will be supplied to each partner institution (200 + 50
extra) for adding them to each collection tube, prior to sampling. More information regarding
PlutoF usage, labels and procedures will be provided further in the document.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Materials
List of materials needed for Malaise trap setting and sample collection
A. Malaise trap (includes trap, plastic and metal stacks, cords and provisional collection tube)
B. Extra cords (IMPORTANT: the cords supplied with the traps are faulty, more to read in the section
“Before sampling”)
C. 500mL sterile collection tubes (see Wide-Mouth LDPE Bottles with Closure link)
D. 96% Ethanol
E. Sticky labels with predefined QR codes (provided by BIOPOLIS-CIBIO)
F. Transparent tape (to provide extra fixation for the sticky labels on the tube)
G. Cable ties
H. Hammer
I. Gray duct tape
J. Extra stacks
Permits and documentation
It is highly important to make sure all the permits and necessary documentation are ready
before sampling. To prevent any delays, check regulation and start processing your permits as
soon as possible.
Permission from local authorities, property owners, rangers, or protected areas stakeholders
can be another factor of potential delay or cancellation. Make sure you formalise the
authorisation to sample on your selected area on time, and if possible, obtain a written
confirmation.
Have copies of any legal documentation ready in case they are needed.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Before starting
Make sure you have all sampling materials organized, to have sorted fieldwork logistics (e.g.,
vehicle, budget, personnel, a fixed day of the week for collection, etc.), to consider habitat
characteristics, sampling location, storing equipment, obtaining all necessary
permissions/authorization for collecting specimens (see previous section).
It is advisable to have more than one site chosen for each altitudinal gradient, when possible, as
a backup site. This is particularly relevant if the sites selected for this project are new for the
team, as is important to consider anthropogenic factors that may disrupt the traps, like hiking
visitors, private landowners, cattle, vandalism, etc.
Malaise traps ordered for the BGE project contain a plastic grid (located in the collection
mechanism in the top of the trap) that comes within the trap. For the High Mountain System
sampling it needs to be removed prior to setting the trap. Otherwise arthropods larger than the
size of the grid will not be sampled. In Figure 3 is shown an example of how to remove the
plastic grid and to secure the mesh back using cable ties.
Figure 3. Example of how to remove the plastic grid included in the Malaise trap.
NOTE: The cords provided with the Malaise traps are showing to be too weak and will probably
give up after normal usage. Please make sure you buy stronger material cords and place them
instead. The extra cords can be useful as spares or to reinforce the structure when necessary.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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To optimize time of sampling and storing, the QR codes stickers should be placed on the sterile
tubes and reinforced with tape prior to sampling. Partner’s coordinators must be registered in
the PlutoF platform (“Become a user”: Register fill in details) in order to have access to the
corresponding project (High Mountain Systems - “name of partner institution”) when using the
PlutoF Go app, to appear as a collector. Alternatively, project coordinators can add “persons”
into the platform (Menu “Persons” Add fill in details) if the collector will be a person that will
be only involved in fieldwork. Make sure you have downloaded the app and enter your personal
data correctly. Further information on PlutoF Go is given on page 13. Support will be provided
whenever needed before, during and after the fieldwork (see contact details at the
end of this document).
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Setting Malaise traps and sample collection
The design of the trap relies on insects being attracted to the highest and brightest part of the
trap. When setting up the trap, ensure that the part that collects the insects (the trap head) is
facing uphill. Ideally, position the trap perpendicular to the path of insect flight, in areas with
minimal undergrowth, such as forest edges, clearings, or elevated locations. Take into account
potential disruptions by wildlife or humans, as well as the direction of the prevailing winds.
Once you have chosen a location, follow the Malaise trap instruction sheet to securely assemble
the trap. A video of how to set up a Malaise trap can be found in this link. When possible, fasten
the front and/or back ropes to nearby trees to provide extra support, and use metal pegs to
attach the bottom rings of the traps to the ground (Figure 4, left), placed in opposite directions to
the trap. Particularly if your sites are located in areas of strong winds, it is advisable to attach
the trap poles to a 1 m - 1.5m stake or post at the highest points to prevent the trap from
toppling over. Use gray tape to reinforce the joints of the trap's metal frame and increase its
stability and resilience (Figure 4, right).
Figure 4. Metal stack/peg placed at one of the extremes of the Malaise trap (left). Gray tape placed
in the joints of the trap metal structure for reinforcement (right).
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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When assembling the trap, make sure to put tension in all the extremes, and that the head
structure in the front (i.e. the tent-like metal structure) is parallel to the tail structure support. To
ensure the entrance is fully open, imagine you are an insect and fly into the trap towards the
trap head (Figure 5), making sure that there is a clear path/entry to the collection tube at the top
of the trap). Check to not over-stretch the mesh, as this will most likely block the path too.
Figure 5. Arthropod view to “the light” path.
Once the trap is set (Figure 6, left), carefully attach the BGE.HMS labeled collection tube tightly
to the trap head, with 96% ethanol (around ¾ would work), and secure it with the white straps
on the trap (Figure 6, right). Take pictures of the trap set and its location. Begin collecting on a
day of the week when you can reliably return for the duration of the sampling period.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Figure 6. Malaise trap set in Serra de Estrela (left). Example of a collection tube correctly placed
and secured in the trap using the white straps around it (right).
Every week, two tubes will be collected per altitudinal site, making a total of 10 tubes per week,
during 20 weeks (200 tubes). Take some extra tubes with you to the field in case something
happens when swapping tubes. Each collection tube should be handled carefully to prevent
contamination. On the collection day, the tube must be topped up with 96% ethanol.
REMEMBER: When collecting the tubes from the traps each week, tubes should be closed and
not opened again.
There could always be problems or eventualities with the Malaise traps (e.g. vandalized, cattle
playing ground, extreme weather). Placing the two Malaise traps away from each other and in
places that cannot be easily seen might help. If something happens to the trap, you can set one
of the replacements nearby, and take a note if the sample was retrieved or lost. Institutional
signs may also help to protect the traps.
After sampling, make sure that all the tubes are well closed and store them at room
temperature. In the PlutoF platform, complete any missing information.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Shipment
For the BGE HMS Arthropods and the Pollinator Communities projects, samples will be shipped
to Dr. Brent Emerson, based in the Institute of Natural Products and Agrobiology, (Instituto de
Productos Naturales y Agrobiología, IPNA-CSIC), in Canary Islands:
Brent Emerson
Island Ecology and Evolution Research Group
Instituto de Productos Naturales y Agrobiología (IPNA-CSIC)
C/Astrofísico Francisco Sánchez 3
La Laguna, Tenerife, Canary Islands, 38206, Spain
Before shipping the samples, make sure that the IPNA-CSI lab has
confirmed the availability to receive the samples (partners will ship
samples in different rounds). Contact details will be provided by
emailing the main contact of this SOP (at the end of the page).
For shipping, It is needed to remove as much ethanol as possible
from each tube, leaving only enough to keep the arthropods
“moist”. To do this, please make sure you are working in a sterile
environment, i.e. laboratory, and open each tube using gloves to
decant the supernatant ethanol. Be careful to not
cross-contaminate any tube.
Full instructions of how to ship Malaise traps tubes are described
in the Malaise traps - Bulk samples Shipping instructions document, please refer to the
information when needed.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Registering samples in PlutoF Go app
PlutoF is an online data management and computing service provider for biological data. PlutoF
Go is the app that will be used to record samples directly in the field. Before using the app, the
data collector should be registered on the PlutoF website. This can be done by the user in the
option Become a user”, or to be added by the BGE project manager directly on her/his
workbench
1
. You can fill all the available information within the Bulk sample option, but it is
required at least to have the data detailed below:
1. Open the PlutoF Go app
2. Go to Add material sample box (If this option is not visible in the main page, go to
Settings, scroll down to Material sample, activate Enable material sample gathering and
make sure the Bulk form is highlighted in green as well).
3. The Location button will show your position in the map. You
should record all the necessary information at the moment of the
sample collection, therefore it should capture the coordinates
detected by your device’s GPS. If there is no internet signal you
can still get the coordinates by pressing the compass icon (top
right inside the map box).
1
The PlutoF project manager will be the only one authorized to add any person to the working project.
Any team member will be automatically notified by email when added to any project.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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4. Start date: insert the collection date (when the tube
is removed from the trap).
5. Project: choose the High Mountain Systems -
Partner option. Partner: Choose the project according to
your institution (compulsory field).
6. Choose form: select “Bulk”.
7. Sample ID: click on the code icon and point your
device camera to the corresponding QR code provided for
the sampling. QR codes are unique and cannot be added
multiple times (compulsory field).
8. Subcode: Add Partner ID, location of gradients/stages +
trap in a consistent way for all the sampling, for example:
Portugal (PT), Stage 1 (S1) (~1000m altitude), Malaise trap a (a) and Malaise trap b (b) = PT.S1a, PT.S1b
PT, Stage 2 (~1300m altitude), Malaise 2a and Malaise 2b (PT.2a, PT.2b). And so on until the last altitudinal
stage (PT.S5a and PT.S5b)
These subcodes will correspond to the same traps during all the sampling period; each BGE.HMS00XX entry
should have these subcodes
9. Description: Write “Placement date of trap or
collection tube in Malaise trap [date]” (i.e. the day when the
Malaise trap was set)
10. Add Collectors: Any person has to be previously
registered/added in the PlutoF platform
11. Add a Trap ID: an identifier for each trap (optional).
12. Add at least one photo of the Malaise trap and its environment
using the panel that is in the top of the Material sample menu.
Use the camera + icon (top left) to generate a picture (see
Figure 5 for an example). Additionally (optional), you can add
any extra information/metadata you think convenient
13. Review all the information submitted is accurate
and click Save at the bottom of the screen
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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14. In the main screen, your entries will be
waiting in the queue to be synchronized. Click on
the cloud icon on the top right to do it.
In the image, there is one good sample entry (dark
letters, intense color) that can be synchronized,
and another entry with missing information (red
letters, faded color) that will not be able to sync
until the missing information is filled. This can be
done by clicking on the entry and revising the info
submitted.
If you cannot sync a sample, click back to that
entry and check your institutional project is
selected. Save, and try to sync again.
Make sure of having an internet connection to sync
your entries, and try to sync often to ensure the
data is saved.
15. You are ready for the next site sampling collection!
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Acknowledgements
Biodiversity Genomics Europe (Grant no.101059492) is funded by Horizon Europe under the
Biodiversity, Circular Economy and Environment call (REA.B.3); co-funded by the Swiss State
Secretariat for Education, Research and Innovation (SERI) under contract numbers 22.00173
and 24.00054; and by the UK Research and Innovation (UKRI) under the Department for
Business, Energy and Industrial Strategy’s Horizon Europe Guarantee Scheme.
References
Abarenkov, K., Tedersoo, L., Nilsson, R. H., Vellak, K., Saar, I., Veldre, V., Parmasto, E., Prous,
M., Aan, A., Ots, M., Kurina, O., Ostonen, I., Jõgeva, J., Halapuu, S., Põldmaa, K., Toots, M.,
Truu, J., Larsson, K-H., and Kõljalg, U. 2010. PlutoF - a web based workbench for ecological
and taxonomic research, with an online implementation for Fungal ITS sequences. Evolutionary
Bioinformatics, 6, 189 - 196.
Hebert, P. D., Cywinska, A., Ball, S. L., and deWaard, J. R. 2003. Biological identifications
through DNA barcodes. Proc Biol Sci. 7,270(1512):313-21.
https://doi.org/10.1098/rspb.2002.2218.
Outhwaite, C. L., McCann, P., and Newbold, T. (2022). Agriculture and climate change are
reshaping insect biodiversity worldwide. Nature, 605(7908):97-102.
https://doi.org/10.1038/s41586-022-04644-x.
Srivathsan, A., Ang, Y., Heraty, J.M., Hwang, W. S., Jusoh W. F. A., Kutty, S. N., Puniamoorthy,
J., Yeo, D., Roslin, T., and Meier, R. (2023). Convergence of dominance and neglect in flying
insect diversity. Nat Ecol Evol. https://doi.org/10.1038/s41559-023-02066-0
Taberlet, P., Coissac, E., Hajibabaei, M., and Rieseberg, L.H. 2012. Environmental DNA.
Molecular Ecology, 21: 1789-1793. https://doi.org/10.1111/J.1365-294x.2012.05542.X.
Young, M. R. and Hebert, P. D. N. 2022. Unearthing soil arthropod diversity through DNA
metabarcoding. PeerJ. 1,10:e12845. https://doi.org/10.7717/peerj.12845.
Useful contact
Laura Nájera Cortazar | Associação Biopolis - CIBIO Centro de Investigação em
Biodiversidade e Recursos Genéticos [BIOPOLIS - CIBIO] [email protected]
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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